Hindawi Publishing Corporation Mediators of Inflammation Volume 2014, Article ID 370530, 12 pages http://dx.doi.org/10.1155/2014/370530 Research Article Chrysophanol Inhibits NALP3 Inflammasome Activation and Ameliorates Cerebral Ischemia/Reperfusion in Mice Nan Zhang,1 Xiangjian Zhang,1,2,3 Xiaoxia Liu,1 Hong Wang,1 Jing Xue,1 Jingying Yu,1 Ning Kang,1 and Xiaolu Wang2 1 Department of Neurology, Second Hospital of Hebei Medical University, Shijiazhuang, Hebei 050000, China 2 Hebei Collaborative Innovation Center for Cardio-Cerebrovascular Disease, Shijiazhuang, Hebei 050000, China 3 Hebei Key Laboratory for Neurology, Shijiazhuang, Hebei 050000, China Correspondence should be addressed to Xiangjian Zhang; [email protected] Received 30 December 2013; Revised 15 March 2014; Accepted 3 April 2014; Published 29 April 2014 Academic Editor: Muzamil Ahmad Copyright © 2014 Nan Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The most effective way to contain cerebral ischemic injury is reperfusion; however, reperfusion itself may result in tissue injury, for which inflammatory damage is one of the main causative factors. NALP3 inflammasome is a multiprotein complex. It consists of NALP3, ASC, and caspase-1, whose function is to switch on the inflammatory process. Chrysophanol is an extract from plants of Rheum genus and it possesses many pharmacological effects including its anti-inflammation activity. In this study, the effects of chrysophanol in cerebral ischemia/reperfusion and the potential mechanisms were investigated. Male CD1 mice were subject to transient middle cerebral artery occlusion (tMCAO). The NALP3 inflammasome activation status and its dynamic expression during the natural inflammatory response induced by tMCAO were first profiled. The neuroprotective effects of chrysophanol were then assessed and the potential mechanisms mediating the observed neuroprotection were then explored. Physical parameters including neurological deficit, infarct size, brain edema, and BBB permeability were measured at 24 h after tMCAO. Confocal microscopy, Western blotting, immunohistochemistry, and qRT-PCR techniques were utilized to analyze the expression of NALP3 inflammasome and IL-1. Our results indicated that the brain tissue damage during cerebral ischemia/reperfusion is accompanied by NALP3 inflammasome activation. Chrysophanol could inhibit the activation of NALP3 inflammasome and protect cerebral ischemic stroke. 1. Introduction imbalance is associated with brain tissue damage. A previous study has demonstrated that mice deficient in IL-1 show Stroke constitutes one of the major causes leading to disability improved survival, reduced the infarct volume, and improved and death worldwide [1]. Inflammatory response has been neurological functions as compared with wild-type mice after confirmed to play a detrimental role in the pathogenesis of middle cerebral artery occlusion (MCAO) [3]. cerebral ischemia/reperfusion (I/R) injury [2]. The relation- Recent evidence suggested that the downstream process- ship between inflammation and cerebral I/R outcome has ing of IL-1 isregulatedbysomecytosolicfactorssuchas assured the considerable and continued interest in the devel- inflammasomes, a family of protein complexes that were opment of anti-inflammation oriented therapies to mitigate recently identified as the cellular machinery responsible I/R-induced brain damage. for recognizing pathogen-associated molecular patterns and The inflammatory process involves activation and inter- reacting to these through activation of inflammatory pro- action of a broad spectrum of immune factors pioneered cesses [4]. Among different types of inflammasomes is the by the upregulation of proinflammatory cytokines. These NALP3 inflammasome, which has been well characterized cytokines, including members of the interleukin (IL)-1 family in a variety of mammalian cells. It is characterized as such as IL-1, are detected in the infarct area and their a proteolytic complex mainly composed of the NACHT 2 Mediators of Inflammation domain-, leucine-rich repeat-, and pyrin domain (PYD)- middle cerebral artery (MCA) was occluded by inserting containing protein 3 (NALP3), the adaptor protein apoptosis- a heparin-dampened monofilament nylon suture (Beijing associated speck-like protein (ASC), and caspase-1 [5]. The Sunbio Biotech Co., Ltd., Beijing, China) into the ICA, NALP3 inflammasome can be activated by bacterial tox- which was moved forward until the flow of right MCA was ins [6] or pathogen-associated molecular patterns, such as reduced to 20%∼30% of basal flow, which was monitored by muramyldipeptide, and other stimuli. NALP3 can also detect a blood flow monitor (moor VMS-LDF, Moor Instruments and respond to endogenous stress-associated danger signals, Ltd., UK). One hour later, the filament was gently withdrawn such as ATP [7], ROS [8], monosodium urate crystals [9], for the reperfusion. Sham-operated mice underwent the same low intracellular potassium concentrations, sodium overload procedures except inserting a filament. The animals’ body ∘ [10], or -amyloid by activating caspase-1, and active caspase- temperature was also monitored and maintained at 36.5 Cto ∘ 1subsequentlymaturestheproinflammatoryIL-1 family 37.5 C during experiment [22–24]. cytokines by cleaving their proforms into biologically active cytokines in turn [11, 12]. Active IL-1 then triggers the IL- 1 receptors on surrounding tissues [13, 14], leading to the 2.3. Experimental Groups and Treatments. All animals were activation of multiple cytokines involved in the inflammation divided into two groups: the control group and the drug cascade, including IL-8, TNF, and IL-17 [15, 16](Figure 1). treatment group. For the control group, a total of 63 mice were However, the role of NALP3 inflammasome in cerebral I/R randomly divided into 7 subgroups, 9 mice each, including inflammatory disorders has not been well explored [17, 18]. the Sham controls and untreated controls for six time points Chrysophanol (CHR), a member of the anthraquinone after tMCAO (3 h, 6 h, 12 h, 24 h, 48 h, and 72 h). For the family, was originally extracted from plants of Rheum genus treatment group, 162 mice were randomly divided into 6 (Figure 2).CHRhasbeenshowntohavemultiplepharma- groups:(1)theShamgroup:animalsreceivedShamoperation cological effects, including its anti-inflammation activity, for and equal volume of 0.9% NaCl; (2) tMCAO group: animals which the underlying mechanisms remain to be elucidated. received tMCAO and equal volume of 0.9% NaCl; (3) Vehicle Previous studies have shown that CHR inhibits caspase-1 and group: animals underwent tMCAO and equal volume of 1% itsdownstreamcytokinesexpressionincolitis[19]. However, DMSO and 1% Tween-80 prepared with 0.9% NaCl; and (4) little is known on the relationship between CHR and NALP3 three CHR groups: animals underwent tMCAO and were inflammasome during cerebral I/R. treated with a high dose of CHR, (10 mg/kg, CHR-H) or Using mouse transient middle cerebral artery occlusion middle dose of CHR (1 mg/kg, CHR-M) and low dose of CHR (tMCAO) model, this study was designed to examine the cel- (0.1mg/kg,CHR-L).Micewerereanesthetizedandsacrificed lular distribution and dynamic expression of NALP3 inflam- at 24 h after tMCAO. The CHR product (Nanjing Zelang masome during cerebral I/R and investigate whether CHR Medical Technological Co., Ltd., Jiangsu, China) with purity has any neuroprotective effect and what are the underlying of more than 98% was first dissolved in 0.9% NaCl including mechanisms mediating this protection if it does have. 1% DMSO and 1% Tween-80 prior to the treatments. Drug or solvent was injected intraperitoneally 30 minutes prior to tMCAO operation. 2. Methods ∼ 2.4. Neurological Deficit Score. The infarct volume, brain ede- 2.1. Experimental Animals. Male CD1 mice (25 30 g) were ma, and blood brain barrier (BBB) permeability were deter- purchased from the Vital River Laboratory Animal Technol- mined by an examiner blinded to the experimental grouping ogy Co., Ltd., Beijing, China. All mice were given at least 2 at 24 h post-tMCAO for the mice from the drug treatment days to acclimatize ahead of any experimentation. During this group. Meanwhile, a neurological test was administered on period, all mice have free access to food and water. Animal these animals following a scoring system adapted from the houses were maintained in a 12/12 h light/dark cycle with ∘ one developed by Longa et al. [20] as follows: 0, no deficits; humidity of 60% ± 5% and ambient temperature of 22 C ± ∘ 1, difficulty in fully extending the contralateral forelimb; 2, 3 C. The experimental procedures were approved by the local unable to extend the contralateral forelimb; 3, mild circling experimental ethics committee as well as the institutional to the contralateral side; 4, severe circling; and 5, falling to animal care and use committee. the contralateral side. 2.2. Animal Surgery for tMCAO. The mouse tMACO model 2.5. Brain Infarct Volume. Infarct volume of brain was mea- was established by following the surgical procedures as pre- sured at 24 h after tMCAO. After the brains (=6in each viously described [20, 21]. In brief, animals were anesthetized group) were dissected, each brain sample was cut into five with chloral hydrate (350 mg/kg, intraperitoneally). Loss of slices with 2 mm thickness, incubated